Process for increasing the stability of a composition comprising polyunsaturated omega-6 fatty acids

ABSTRACT

A processes for increasing the stability of a composition containing polyunsaturated an omega-6 fatty acid against oxidation proceeds by (i) providing a starting composition containing at least one polyunsaturated omega-6 fatty acid component; (ii) providing a lysine composition; (iii) admixing an aqueous, an aqueous-alcoholic or an alcoholic solution of starting composition and lysine composition, and subjecting a resulting admixture to spray drying conditions subsequently, thus forming a solid product composition containing at least one salt of a cation derived from lysine with an anion derived from the polyunsaturated omega-6 fatty acid; the product composition exhibiting a solvent content SC selected from the following; SC&lt;5 wt %, SC&lt;3 wt %, SC&lt;1 wt %, or &lt;0.5 wt %.

Numerous health benefits have been correlated with the supplementalintake of polyunsaturated fatty acids (PUFAs) by an extensive body ofevidence gathered over the course of the past several decades.Prevention of cardiovascular disease and reducing the symptoms ofinflammatory conditions are amongst the most prominent examples,however, preventing the promotion and progression stages of some typesof cancer, reducing blood pressure and blood cholesterol as well aspositive effects in the treatment of depression and schizophrenia,Alzheimer's disease, dyslexia, and attention-deficit or hyperactivitydisorder, amongst others, have been reported as well. Furthermore,because some PUFAs are considered to be essential for the development ofbrain, nervous system and eye, nowadays routinely, infant nutrition issupplemented with specific PUFAs.

However, the manufacture of food, nutritional and pharmaceuticalproducts containing PUFAs is impeded by their high susceptibilitytowards oxidative deterioration. Oxidation has negative, bothnutritional and organoleptic, consequences; namely, changes innutritional value such as the destruction of essential fatty acids;rancidity which produces off-flavors and pronounced odors; color changessuch as darkening of fats and oils, as well as flavor loss. Oxidation ofPUFAs produces a complex mixture of volatile secondary oxidationproducts, and these cause particularly objectionable off-flavors.

Three different strategies have been described in the art and applied inindustry to stabilize PUFAs against oxidative deterioration(Arab-Tehrany E. et al., Trends in Food Science & Technology 25 (2012)24-33):

-   -   addition of antioxidants,    -   microencapsulation, and    -   modified atmosphere packaging.

Despite the fact that these strategies offer solutions for a number ofoxidation-related problems, new approaches are still needed in order torespond to remaining challenges in current scenarios and potentialfuture settings dictating particular technical and economic boundaryconditions.

It was now found that compositions comprising polyunsaturated omega-6fatty acids can be stabilized against oxidation by a process comprisingthe following steps:

-   -   (i) providing a starting composition comprising at least one        polyunsaturated omega-6 fatty acid component;    -   (ii) providing a lysine composition;    -   (iii) admixing aqueous, aqueous-alcoholic or alcoholic solutions        of starting composition and lysine composition, and subjecting        resulting admixture to spray drying conditions subsequently,        thus forming a solid product composition comprising at least one        salt of a cation derived from lysine with an anion derived from        a polyunsaturated omega-6 fatty acid; the product composition        exhibiting a solvent content SC selected from the following;        SC<5 wt %, SC<3 wt %, SC<1 wt %, SC<0.5 wt %.

In the context of the present invention the term PUFA is usedinterchangeably with the term polyunsaturated fatty acid and defined asfollows: Fatty acids are classified based on the length and saturationcharacteristics of the carbon chain. Short chain fatty acids have 2 toabout 6 carbons and are typically saturated. Medium chain fatty acidshave from about 6 to about 14 carbons and are also typically saturated.Long chain fatty acids have from 16 to 24 or more carbons and may besaturated or unsaturated. In longer chain fatty acids there may be oneor more points of unsaturation, giving rise to the terms“monounsaturated” and “polyunsaturated,” respectively. In the context ofthe present invention long chain polyunsaturated fatty acids having 20or more carbon atoms are designated as polyunsaturated fatty acids orPUFAs.

PUFAs are categorized according to the number and position of doublebonds in the fatty acids according to well established nomenclature.There are two main series or families of LC-PUFAs, depending on theposition of the double bond closest to the methyl end of the fatty acid:The omega-3 series contains a double bond at the third carbon, while theomega-6 series has no double bond until the sixth carbon. Thus,docosahexaenoic acid (“DHA”) has a chain length of 22 carbons with 6double bonds beginning with the third carbon from the methyl end and isdesignated “22:6 n-3” (all-cis-4,7,10,13,16,19-docosahexaenoic acid).Another important omega-3 PUFA is eicosapentaenoic acid (“EPA”) which isdesignated “20:5 n-3” (all-cis-5,8,11,14,17-eicosapentaenoic acid). Animportant omega-6 PUFA is arachidonic acid (“ARA”) which is designated“20:4 n-6” (all-cis-5,8,11,14-eicosatetraenoic acid).

Other Omega-3 PUFAs Include:

Eicosatrienoic acid (ETE) 20:3 (n-3) (all-cis-11,14,17-eicosatrienoicacid), Eicosatetraenoic acid (ETA) 20:4 (n-3)(all-cis-8,11,14,17-eicosatetraenoic acid), Heneicosapentaenoic acid(HPA) 21:5 (n-3) (all-cis-6,9,12,15,18-heneicosapentaenoic acid),Docosapentaenoic acid (Clupanodonic acid) (DPA) 22:5 (n-3)(all-cis-7,10,13,16,19-docosapentaenoic acid), Tetracosapentaenoic acid24:5 (n-3) (all-cis-9,12,15,18,21-tetracosapentaenoic acid),Tetracosahexaenoic acid (Nisinic acid) 24:6 (n-3)(all-cis-6,9,12,15,18,21-tetracosahexaenoic acid).

Other Omega-6 PUFAs Include:

Eicosadienoic acid 20:2 (n-6) (all-cis-11,14-eicosadienoic acid),Dihomo-gamma-linolenic acid (DGLA) 20:3 (n-6)(all-cis-8,11,14-eicosatrienoic acid), Docosadienoic acid 22:2 (n-6)(all-cis-13,16-docosadienoic acid), Adrenic acid 22:4 (n-6)(all-cis-7,10,13,16-docosatetraenoic acid), Docosapentaenoic acid(Osbond acid) 22:5 (n-6) (all-cis-4,7,10,13,16-docosapentaenoic acid),Tetracosatetraenoic acid 24:4 (n-6)(all-cis-9,12,15,18-tetracosatetraenoic acid), Tetracosapentaenoic acid24:5 (n-6) (all-cis-6,9,12,15,18-tetracosapentaenoic acid).

The preferred omega-6 PUFAs used in the embodiments of the presentinvention are arachidonic acid (“ARA”) and gamma-linolenic acid (“GLA”).

Without wanting to be bound by theory it appears that the increasedstability towards oxidation achieved by the process of the presentinvention is a result of salt formation between lysine and PUFA.Corresponding stability increase is not observed for PUFAs that remainfree acids, or form part of an ester or an inorganic salt, e.g. withNa+, K+, Ca2+, or Mg2+.

Compositions comprising polyunsaturated omega-6 fatty acids that can bestabilized against oxidation by the process of the present invention maybe any compositions containing substantial amounts of freepolyunsaturated omega-6 fatty acids. Such compositions may furthercomprise other naturally occurring fatty acids in free form. Inaddition, such compositions may further comprise constituents that bythemselves are solid, liquid or gaseous at room temperature and standardatmospheric pressure. Corresponding liquid constituents includeconstituents that can easily be removed by evaporation and could thus beconsidered as volatile constituents as well as constituents that aredifficult to remove by evaporation and could thus be considered asnon-volatile constituents. In the present context gaseous constituentsare considered as volatile constituents. Typical volatile constituentsare water, alcohols and supercritical carbon dioxide.

Compositions comprising polyunsaturated omega-6 fatty acids that can bestabilized against oxidation by the process of the present invention maybe obtained from any suitable source material which, additionally, mayhave been processed by any suitable method of processing such sourcematerial. Typical source materials include animal-derived products suchas eggs, but in particular, material derived from microbial, especiallyfungal and/or algal fermentation. Typically, such material furthercontains substantial amounts of other naturally occurring fatty acids.Typical methods of processing such source materials may include stepsfor obtaining crude oils such as extraction and separation of the sourcematerial, as well as steps for refining crude oils such as settling anddegumming, de-acidification, bleaching, and deodorization, and furthersteps for producing omega-6 PUFA-concentrates from refined oils such asde-acidification, trans-esterification, concentration, and deodorization(cf. e.g. EFSA Scientific Opinion on Fish oil for Human Consumption).Any processing of source materials may further include steps for atleast partially transforming omega-6 PUFA-esters into the correspondingfree omega-6 PUFAs or inorganic salts thereof.

Preferred compositions comprising polyunsaturated omega-6 fatty acidsthat can be stabilized against oxidation by the process of the presentinvention can be obtained from compositions mainly consisting of estersof omega-6 PUFAs and other naturally occurring fatty acids by cleavageof the ester bonds and subsequent removal of the alcohols previouslybound as esters. Preferably, ester cleavage is performed under basicconditions. Methods for ester cleavage are well known in the art.

In the context of the present invention stabilizing compositions againstoxidation means that the stability of such compositions towardsoxidation is increased. One measure for quantifying the stability of acomposition towards oxidation is the induction time in a Rancimat test.Protocols for performing the Rancimat test are well known in the artand/or provided by manufacturers of instruments used for performing theRancimat test. An alternative measure for the stability of a compositiontowards oxidation can be obtained as follows: The stability of two ormore samples of any composition or compound towards oxidation can becompared by (1) initially measuring the degree of oxidation of thesamples, followed by (2) subjecting the samples to comparable(oxidizing) conditions and (3) measuring the degree of oxidation of thesamples thereafter. The sample with the smallest increase of its degreeof oxidation exhibits the highest stability towards oxidation under thegiven conditions, whereas the sample with the largest increase of itsdegree of oxidation exhibits the lowest stability towards oxidationunder the given conditions. Increase of the degree of oxidation of asample can be expressed in absolute terms, i.e. as the difference of thevalues obtained before and after subjecting to oxidizing conditions, or,alternatively, the increase can be expressed in relative terms, i.e. asthe ratio of the values obtained before and after subjecting tooxidizing conditions. Evidently, a decrease of the degree of oxidationresulting from exposure to oxidizing conditions indicates a very highlevel of stability towards oxidation which should be interpreted asbeing even higher than the level of stability of a sample yielding anunchanged degree of oxidation as a result of exposing the sample tocomparable oxidizing conditions.

Several measures are known in the art for quantifying the degree ofoxidation of a sample. In the broadest sense of the present invention,any of these measures can be used. In preferred embodiments of thepresent invention one or more of the following measures are used forquantifying the degree of oxidation: Peroxide Value (PV), AnisidineValue (AV), Totox Value. PV is a measure of primary oxidation products(hydroperoxide-formation at double bonds) and AV is a measure ofsecondary degradation products (carbonyl compounds). Totox Value iscalculated as Totox=2*PV+AV (wherein PV is specified in milliequivalents02 per kg of sample). Procedures for determining Peroxide Value (PV) andAnisidine Value (AV) have been described in the literature (cf. e.g.Official Methods and Recommended Practices of the AOCS, 6^(th) Edition2013, Edited by David Firestone, ISBN 978-1-893997-74-5; or, e.g. PV canbe determined according to Ph. Eur. 2.5.5 (01/2008:20505), AV can bedetermined according to Ph. Eur. 2.5.36 (01/2008:20536)).

An exemplary procedure for determining the Peroxide Value (PV) of asample is performed as follows:

Reagents and Solution:

1. Acetic Acid—chloroform solution (7.2 ml Acetic Acid and 4.8 mlChloroform).

2. Saturated Potassium Iodide solution. Store in the dark.

3. Sodium thiosulfate solution, 0.1N. Commercially available.

4. 1% Starch solution. Commercially available.

5. Distilled or deionized water.

Procedure:

Conduct a blank determination of the reagents.

1. Weigh 2.00 (±0.02) g of sample into a 100 ml glass stopperedErlenmeyer flask. Record weight to the nearest 0.01 g.

2. By graduated cylinder, add 12 ml of the acetic acid—chloroformsolution.

3. Swirl the flask until the sample is completely dissolved (carefulwarming on a hot plate may be necessary).

4. Using 1 ml Mohr pipette, add 0.2 ml of saturated potassium iodidesolution.

5. Stopper the flask and swirl the contents of the flask for exactly oneminute.

6. Immediately add by graduated cylinder, 12 ml of either distilled ordeionized water, stopper and shake vigorously to liberate the iodinefrom the chloroform layer.

7. Fill the burette with 0.1N sodium thiosulfate.

8. If the starting color of the solution is deep red orange, titrateslowly with mixing until the color lightens. If the solution isinitially a light amber color, go to step 9.

9. Using a dispensing device, add 1 ml of starch solution as indicator.

10. Titrate until the blue gray color disappears in the aqueous (upperlayer).

11. Accurately record the mls of titrant used to two decimal places.

Calculation:

S=titration of sample

B=titration of blank

Peroxide value=(S−B)*N thiosulfate*1000/weight of sample

An exemplary procedure for determining the Anisidine Value (AV) of asample is performed as follows:

The anisidine value is defined as 100 times the optical density measuredin a 1 cm cell of a solution containing 1 g of the substance to beexamined in 100 ml of a mixture of solvents and reagents according tothe following method. Carry out the operations as rapidly as possible,avoiding exposure to actinic light.

Test solution (a): Dissolve 0.500 g of the substance to be examined intrimethylpentane and dilute to 25.0 ml with the same solvent.

Test solution (b): To 5.0 ml of test solution (a) add 1.0 ml of a 2.5g/I solution of p-anisidine in glacial acetic acid, shake and storeprotected from light.

Reference solution: To 5.0 ml of trimethylpentane add 1.0 ml of a 2.5g/I solution of p-anisidine in glacial acetic acid, shake and storeprotected from light. Measure the absorbance of test solution (a) at themaximum at 350 nm using trimethylpentane as the compensation liquid.Measure the absorbance of test solution (b) at 350 nm exactly 10 minafter its preparation, using the reference solution as the compensationliquid. Calculate the anisidine value (AV) from the expression:

AV=(25*(1.2*A1−A2))/m

A1=absorbance of test solution (b) at 350 nm,

A2=absorbance of test solution (a) at 350 nm,

m=mass of the substance to be examined in test solution (a), in grams.

When comparing the stability of samples towards oxidation by (1)measuring the degree of oxidation, (2) subjecting to oxidizingconditions, and (3) measuring the degree of oxidation again, in thecontext of the present invention, preferably, the degree of oxidation insteps (1) and (3) is assessed by determining Peroxide Value (PV) and/orAnisidine Value (AV); further, preferably, the oxidizing conditions instep (2) are selected from one of the following: storage in opencontainers exposed to air at room temperature over a defined period oftime of at least ten days; storage in open containers exposed to air at50° C. over a defined period of time of at least three days.

In the context of the present invention increasing the stability of acomposition towards oxidation by a process means that at least onemeasure describing the stability of a composition towards oxidation,e.g. at least one measure as described above, is increased after thecomposition is subjected to the process.

In the context of the present invention starting compositions comprisingat least one polyunsaturated omega-6 fatty acid component may be anycompositions containing substantial amounts of at least onepolyunsaturated omega-6 fatty acid component, wherein each type (i.e.molecular species) of free omega-6 PUFA (with “free” indicating thepresence of a free carboxylic acid function) constitutes a differentpolyunsaturated omega-6 fatty acid component. Such compositions mayfurther comprise other naturally occurring fatty acids in free form. Inaddition, such compositions may further comprise constituents that bythemselves are solid, liquid or gaseous at room temperature and standardatmospheric pressure. Corresponding liquid constituents includeconstituents that can easily be removed by evaporation and could thus beconsidered as volatile constituents as well as constituents that aredifficult to remove by evaporation and could thus be considered asnon-volatile constituents. In the present context gaseous constituentsare considered as volatile constituents. Typical volatile constituentsare water, alcohols and supercritical carbon dioxide.

Accordingly, typical starting compositions, without taking account forvolatile constituents, have a PUFA-content PC (i.e. the total content ofone or more free polyunsaturated omega-6 fatty acids) of at least 15 wt%, up to 85 wt % of other naturally occurring fatty acids in free form,and up to 5 wt % of other constituents that by themselves are solid orliquid at room temperature and standard atmospheric pressure. However,higher grades of polyunsaturated omega-6 fatty acids can be obtained bypurification of the respective starting materials. In a preferredembodiment of the present invention starting compositions, withouttaking account for volatile constituents, have a PUFA-content PC (i.e.the total content of one or more free polyunsaturated omega-6 fattyacids) of at least 30 wt %, up to 70 wt % of other naturally occurringfatty acids in free form, and up to 5 wt % of other constituents that bythemselves are solid or liquid at room temperature and standardatmospheric pressure. In another preferred embodiment of the presentinvention starting compositions, without taking account for volatileconstituents, have a PUFA-content PC (i.e. the total content of one ormore free polyunsaturated omega-6 fatty acids) of at least 50 wt %, upto 50 wt % of other naturally occurring fatty acids in free form, and upto 5 wt % of other constituents that by themselves are solid or liquidat room temperature and standard atmospheric pressure. In anotherpreferred embodiment of the present invention starting compositions,without taking account for volatile constituents, have a PUFA-content PC(i.e. the total content of one or more free polyunsaturated omega-6fatty acids) of at least 75 wt %, up to 25 wt % of other naturallyoccurring fatty acids in free form, and up to 5 wt % of otherconstituents that by themselves are solid or liquid at room temperatureand standard atmospheric pressure. In another preferred embodiment ofthe present invention starting compositions, without taking account forvolatile constituents, have a PUFA-content PC (i.e. the total content ofone or more free polyunsaturated omega-6 fatty acids) of at least 75 wt%, up to 25 wt % of other naturally occurring fatty acids in free form,and up to 1 wt % of other constituents that by themselves are solid orliquid at room temperature and standard atmospheric pressure.

The lysine composition provided in step (ii) of the process of thepresent invention is a composition comprising substantial amounts offree Lysine (Lys). The lysine composition may further compriseconstituents that by themselves are solid, liquid or gaseous at roomtemperature and standard atmospheric pressure. Corresponding liquidconstituents include constituents that can easily be removed byevaporation and could thus be considered as volatile constituents aswell as constituents that are difficult to remove by evaporation andcould thus be considered as non-volatile constituents. In the presentcontext gaseous constituents are considered as volatile constituents.Typical volatile constituents are water, alcohols and supercriticalcarbon dioxide. Typical lysine compositions contain at least 95 wt %, 97wt %, 98 wt %, or 99 wt % of free lysine, without taking account forvolatile constituents. Preferred lysine compositions contain at least 98wt % of free lysine, without taking account for volatile constituents.

In preferred embodiments of the present invention, without accountingfor volatile constituents, starting compositions contain mostly freePUFAs and other naturally occurring fatty acids in free form and lysinecompositions contain mostly free lysine, thus yielding productcompositions mostly consisting of salts of lysine with PUFAs and othernaturally occurring fatty acids. Accordingly, in preferred embodimentsof the present invention, starting composition in step (i) and lysinecomposition in step (ii) are provided in such a manner that at least spwt % of the product composition consist of one or more salts of cationsderived from lysine with anions derived from one or more polyunsaturatedomega-6 fatty acids and other naturally occurring fatty acids, whereinsp is selected from 90, 95, 97, 98, 99, 100.

In step (iii) of the process of the present invention startingcomposition and lysine composition are combined. Combining can beachieved by any means allowing formation of a product compositioncomprising at least one salt of a cation derived from lysine with ananion derived from a polyunsaturated omega-6 fatty acid. Accordingly, atypical way of combining starting composition and lysine compositionwould be admixing aqueous, aqueous-alcoholic or alcoholic solutions ofeach and removing the solvent subsequently. Alternatively, depending onthe remaining constituents of starting composition and lysinecomposition, it may not be necessary to add solvents but could besufficient to combine starting composition and lysine compositiondirectly. In the context of the present invention a preferred way ofcombining starting composition and lysine composition is admixingaqueous, aqueous-alcoholic or alcoholic solutions of each and removingthe solvent subsequently.

In the context of the present invention a cation derived from lysine isa cation obtained by protonation of lysine.

In the context of the present invention an anion derived from apolyunsaturated omega-6 fatty acid is an anion obtained by deprotonationof a polyunsaturated omega-6 fatty acid.

It should be noted that salts of lysine with polyunsaturated fatty acidsper se were known in the art (cf. EP 0734373 B1), however, it wasunknown that such salts exhibit higher stability towards oxidativedegradation as compared to free PUFAs or PUFA-esters.

In view of the intrinsic stability of the salts of lysine with omega-6PUFAs it is unnecessary to add substantial amounts of antioxidants tothese salts. Accordingly, in preferred embodiments of the presentinvention the product composition obtained in step (iii) contains nosubstantial amounts of antioxidants, wherein no substantial amountsmeans that this composition contains less than 5 wt %, 3 wt %, 1 wt %,or 0.1 wt % of antioxidants. In further preferred embodiments theproduct composition contains no antioxidants at all. In preferredembodiments of the present invention the product composition contains nosubstantial amounts of antioxidants, wherein no substantial amountsmeans that the product composition contains less than 5 wt %, 3 wt %, 1wt %, or 0.1 wt % of antioxidants and wherein the antioxidants areselected from vitamin C and esters thereof, erythorbic acid and estersthereof, vitamin E and esters thereof, polyphenols and esters thereof,carotinoids, gallates and esters thereof, butylated hydroxyanisole andesters thereof, butylated hydroxytoluene and esters thereof, rosemaryoil, hexylresorcinol and esters thereof. In further preferredembodiments the product composition contains no antioxidants at all,wherein the antioxidants are selected from vitamin C and esters thereof,erythorbic acid and esters thereof, vitamin E and esters thereof,polyphenols and esters thereof, carotinoids, gallates and estersthereof, butylated hydroxyanisole and esters thereof, butylatedhydroxytoluene and esters thereof, rosemary oil, hexylresorcinol andesters thereof.

According to the invention the product composition exhibits higherstability towards oxidation than the starting composition. This meansthat at least one measure describing the stability of a compositiontowards oxidation, e.g. at least one measure as described above,indicates a higher stability towards oxidation for the productcomposition than for the starting composition.

In preferred embodiments of the present invention free carboxylic acidfunctions and lysine are provided in roughly equimolar quantities inorder to facilitate quantitative salt formation. Accordingly, in apreferred embodiment in the process of the present invention lysinecomposition in step (ii) is provided in such a manner that the ratioR=n(ca)/n(lys) of the amount of carboxylic acid functions n(ca) in thestarting composition provided in step (i) and the total amount of freelysine n(lys) in the lysine composition provided in step (ii) is in arange selected from 0.9<R<1.1, 0.95<R<1.05, 0.98<R<1.02. In aparticularly preferred embodiment R is in the range 0.98<R<1.02. Theamount of carboxylic acid functions n(ca) in the starting compositionprovided in step (i) can be determined by standard analytical procedureswell known in the art, e.g. acid base titration.

In preferred embodiments of the present invention the startingcomposition provided in step (i), does not contain substantial amountsof fatty acid esters, thus yielding a product composition devoid ofsubstantial amounts of fatty acid esters as well. Accordingly, inpreferred embodiments of the present invention the starting compositionprovided in step (i) does not contain more than x(fe) wt % of fatty acidesters, thus yielding a product composition comprising a maximum ofx(fe) wt % of fatty acid esters, wherein x(fe) is selected from 5, 3, 1,0.3, 0. In particularly preferred embodiments x(fe) is 1.

As noted above, salts of lysine with polyunsaturated fatty acids per sewere known in the art (cf. EP 0734373 B1), however, it was unknown thatsuch salts exhibit higher stability towards oxidative degradation ascompared to free PUFAs or PUFA-esters. Importantly, further, lysine-PUFAsalts were described as “very thick transparent oils, which transforminto solids of waxy appearance and consistency at low temperatures” (cf.EP 0734373 B1, page 1, lines 47 to 48). As a result, a person of skillin the art could not have expected that salts of lysine with omega-6PUFAs could be obtained via spray drying procedures. Instead, a personof skill would have expected that such salts would (a) deteriorate underspray drying conditions due to oxidative damage under elevatedtemperatures in the absence of substantial amounts of solvents,antioxidants and protective coatings, and (b) agglomerate into clumpsmechanically prohibitive to the process of spray drying in view of thepresumed appearance of such salts as waxy solids. It is thereforeremarkable that, presently, it was found that salts of lysine withomega-6 PUFAs can in fact be obtained via spray drying in a facilemanner. Conditions for spray drying, always, have to be adapted to theparticular spray-drying equipment used. However, it is well within thescope of routine laboratory work of a person of skill in the art toperform such adaption in the present case.

In order to perform the spray drying step according to the process ofthe present invention aqueous, aqueous-alcoholic or alcoholic solutionsare used. It was found that Lys-salts of PUFAs dissolve poorly in neatalcoholic solvents. It was, further, found that such salts exhibit gellike appearance when dissolved at high concentration in neat water.Aqueous-alcoholic solvent systems may thus be employed for avoiding suchproblems. Accordingly, in preferred embodiments of the present inventionthe solvent of the admixture subjected to spray drying conditions is anaqueous-alcoholic solvent system containing 20 wt % to 90 wt % water and80 wt % to 10 wt % alcoholic solvents.

The solvent content of the solid product composition will vary dependingon spray drying conditions and substrates used, however, it waspresently found that even at very low solvent contents in the solidproduct composition oxidative damage does not occur. As outlined furtherabove, this could not have been expected. Preferably, thus, according tothe present invention a solid product composition with a low solventcontent is obtained. Thus, according to the present invention in step(iii) aqueous, aqueous-alcoholic or alcoholic solutions of startingcomposition and lysine composition are first admixed, and subjected tospray drying conditions subsequently, thus yielding a solid productcomposition comprising at least one salt of a cation derived from lysinewith an anion derived from a polyunsaturated omega-6 fatty acid, with asolvent content SC selected from the following; SC<5 wt %, SC<3 wt %,SC<1 wt %, SC<0.5 wt %. In a particularly preferred embodiment of thepresent invention SC is selected as SC<1 wt %.

The present invention, further, comprises compositions obtainable by anyof the processes of the invention.

The present invention, further, comprises use of compositions,obtainable by any of the processes of the invention, for the manufactureof food products comprising polyunsaturated omega-6 fatty acids.

In the context of the present invention food products comprise but arenot limited to baked goods, vitamin supplements, diet supplements,powdered drinks, doughs, batters, baked food items including e.g. cakes,cheesecakes, pies, cupcakes, cookies, bars, breads, rolls, biscuits,muffins, pastries, scones, and croutons; liquid food products e.g.beverages, energy drinks, infant formula, liquid meals, fruit juices,multivitamin syrups, meal replacers, medicinal foods, and syrups;semi-solid food products such as baby food, yogurt, cheese, cereal,pancake mixes; food bars including energy bars; processed meats; icecreams; frozen desserts; frozen yogurts; waffle mixes; salad dressings;and replacement egg mixes; and further cookies, crackers, sweet goods,snacks, pies, granola/snack bars, and toaster pastries; salted snackssuch as potato chips, corn chips, tortilla chips, extruded snacks,popcorn, pretzels, potato crisps, and nuts; specialty snacks such asdips, dried fruit snacks, meat snacks, pork rinds, health food bars andrice/corn cakes; confectionary snacks such as candy; instant foodproducts, such as instant noodles, instant soup cubes or granulates.

The present invention, further, comprises use of compositions,obtainable by any of the processes of the invention, for the manufactureof nutritional products comprising polyunsaturated omega-6 fatty acids.

In the context of the present invention nutritional products compriseany type of nutraceutical, nutrient or dietary supplement, e.g. forsupplementing vitamins, minerals, fiber, fatty acids, or amino acids.

The present invention, further, comprises use of compositions,obtainable by any of the processes of the invention, for the manufactureof pharmaceutical products comprising polyunsaturated omega-6 fattyacids.

Preferred processes of the present invention are characterized by onethe following selections:

-   -   0.90<R<1.10, x(fe)=5; PC=15; SC<1 wt %    -   0.90<R<1.10, x(fe)=3; PC=15; SC<1 wt %    -   0.90<R<1.10, x(fe)=2; PC=15; SC<1 wt %    -   0.90<R<1.10, x(fe)=1; PC=15; SC<1 wt %    -   0.95<R<1.05, x(fe)=5; PC=15; SC<1 wt %    -   0.95<R<1.05, x(fe)=3; PC=15; SC<1 wt %    -   0.95<R<1.05, x(fe)=2; PC=15; SC<1 wt %    -   0.95<R<1.05, x(fe)=1; PC=15; SC<1 wt %    -   0.98<R<1.02, x(fe)=5; PC=15; SC<1 wt %    -   0.98<R<1.02, x(fe)=3; PC=15; SC<1 wt %    -   0.98<R<1.02, x(fe)=2; PC=15; SC<1 wt %    -   0.98<R<1.02, x(fe)=1; PC=15; SC<1 wt %    -   0.90<R<1.10, x(fe)=5; PC=30; SC<1 wt %    -   0.90<R<1.10, x(fe)=3; PC=30; SC<1 wt %    -   0.90<R<1.10, x(fe)=2; PC=30; SC<1 wt %    -   0.90<R<1.10, x(fe)=1; PC=30; SC<1 wt %    -   0.95<R<1.05, x(fe)=5; PC=30; SC<1 wt %    -   0.95<R<1.05, x(fe)=3; PC=30; SC<1 wt %    -   0.95<R<1.05, x(fe)=2; PC=30; SC<1 wt %    -   0.95<R<1.05, x(fe)=1; PC=30; SC<1 wt %    -   0.98<R<1.02, x(fe)=5; PC=30; SC<1 wt %    -   0.98<R<1.02, x(fe)=3; PC=30; SC<1 wt %    -   0.98<R<1.02, x(fe)=2; PC=30; SC<1 wt %    -   0.98<R<1.02, x(fe)=1; PC=30; SC<1 wt %    -   0.90<R<1.10, x(fe)=5; PC=50; SC<1 wt %    -   0.90<R<1.10, x(fe)=3; PC=50; SC<1 wt %    -   0.90<R<1.10, x(fe)=2; PC=50; SC<1 wt %    -   0.90<R<1.10, x(fe)=1; PC=50; SC<1 wt %    -   0.95<R<1.05, x(fe)=5; PC=50; SC<1 wt %    -   0.95<R<1.05, x(fe)=3; PC=50; SC<1 wt %    -   0.95<R<1.05, x(fe)=2; PC=50; SC<1 wt %    -   0.95<R<1.05, x(fe)=1; PC=50; SC<1 wt %    -   0.98<R<1.02, x(fe)=5; PC=50; SC<1 wt %    -   0.98<R<1.02, x(fe)=3; PC=50; SC<1 wt %    -   0.98<R<1.02, x(fe)=2; PC=50; SC<1 wt %    -   0.98<R<1.02, x(fe)=1; PC=50; SC<1 wt %    -   0.90<R<1.10, x(fe)=5; PC=75; SC<1 wt %    -   0.90<R<1.10, x(fe)=3; PC=75; SC<1 wt %    -   0.90<R<1.10, x(fe)=2; PC=75; SC<1 wt %    -   0.90<R<1.10, x(fe)=1; PC=75; SC<1 wt %    -   0.95<R<1.05, x(fe)=5; PC=75; SC<1 wt %    -   0.95<R<1.05, x(fe)=3; PC=75; SC<1 wt %    -   0.95<R<1.05, x(fe)=2; PC=75; SC<1 wt %    -   0.95<R<1.05, x(fe)=1; PC=75; SC<1 wt %    -   0.98<R<1.02, x(fe)=5; PC=75; SC<1 wt %    -   0.98<R<1.02, x(fe)=3; PC=75; SC<1 wt %    -   0.98<R<1.02, x(fe)=2; PC=75; SC<1 wt %    -   0.98<R<1.02, x(fe)=1; PC=75; SC<1 wt %

Preferred compositions obtainable by a process of the inventionutilizing spray drying in step (iii) as disclosed in the specificationare characterized by one the following selections:

-   -   0.90<R<1.10, x(fe)=1, SC<3 wt %, sp=90    -   0.90<R<1.10, x(fe)=1, SC<3 wt %, sp=95    -   0.90<R<1.10, x(fe)=3, SC<3 wt %, sp=90    -   0.90<R<1.10, x(fe)=3, SC<3 wt %, sp=95    -   0.95<R<1.05, x(fe)=1, SC<3 wt %, sp=90    -   0.95<R<1.05, x(fe)=1, SC<3 wt %, sp=95    -   0.95<R<1.05, x(fe)=3, SC<3 wt %, sp=90    -   0.95<R<1.05, x(fe)=3, SC<3 wt %, sp=95    -   0.98<R<1.02, x(fe)=1, SC<3 wt %, sp=95    -   0.98<R<1.02, x(fe)=1, SC<3 wt %, sp=97    -   0.98<R<1.02, x(fe)=3, SC<3 wt %, sp=95    -   0.98<R<1.02, x(fe)=3, SC<3 wt %, sp=97    -   0.90<R<1.10, x(fe)=1, SC<1 wt %, sp=90    -   0.90<R<1.10, x(fe)=1, SC<1 wt %, sp=95    -   0.90<R<1.10, x(fe)=3, SC<1 wt %, sp=90    -   0.90<R<1.10, x(fe)=3, SC<1 wt %, sp=95    -   0.95<R<1.05, x(fe)=1, SC<1 wt %, sp=90    -   0.95<R<1.05, x(fe)=1, SC<1 wt %, sp=95    -   0.95<R<1.05, x(fe)=3, SC<1 wt %, sp=90    -   0.95<R<1.05, x(fe)=3, SC<1 wt %, sp=95    -   0.98<R<1.02, x(fe)=1, SC<1 wt %, sp=95    -   0.98<R<1.02, x(fe)=1, SC<1 wt %, sp=97    -   0.98<R<1.02, x(fe)=3, SC<1 wt %, sp=95    -   0.98<R<1.02, x(fe)=3, SC<1 wt %, sp=97    -   0.90<R<1.10, x(fe)=1, SC<0.5 wt %, sp=90    -   0.90<R<1.10, x(fe)=1, SC<0.5 wt %, sp=95    -   0.90<R<1.10, x(fe)=3, SC<0.5 wt %, sp=90    -   0.90<R<1.10, x(fe)=3, SC<0.5 wt %, sp=95    -   0.95<R<1.05, x(fe)=1, SC<0.5 wt %, sp=90    -   0.95<R<1.05, x(fe)=1, SC<0.5 wt %, sp=95    -   0.95<R<1.05, x(fe)=3, SC<0.5 wt %, sp=90    -   0.95<R<1.05, x(fe)=3, SC<0.5 wt %, sp=95    -   0.98<R<1.02, x(fe)=1, SC<0.5 wt %, sp=95    -   0.98<R<1.02, x(fe)=1, SC<0.5 wt %, sp=97    -   0.98<R<1.02, x(fe)=3, SC<0.5 wt %, sp=95    -   0.98<R<1.02, x(fe)=3, SC<0.5 wt %, sp=97

Experiments Analytical Methods:

Primary oxidation products (hydroperoxides at double bonds) werequantified by determining the Peroxide Value (PV) according to Ph. Eur.2.5.5 (01/2008:20505). Secondary oxidation products (carbonyl compounds)were quantified by determining the Anisidine Value (AV) according to Ph.Eur. 2.5.36 (01/2008:20536).

Oligomeric PUFA constituents as well as their derivatives (collectivelyreferred to as oligomer content) were quantified by gel-chromatographicmeans (GPC, styroldivinylbenzene-phase with tetrahydrofuran containingtrifluoroacetic acid used as eluent). A refractive index (RI) detectorwas used for detection. Due to the fact that specific response factorsof the constituents of the samples were unknown, proportions werecalculated based upon fractional proportions of the total area ofchromatograms.

Water content was determined by Karl-Fischer titration.

Ethanol content was determined by 1-H-NMR spectroscopy.

Acid values were determined by titration with potassium hydroxide.

Experiment 1: Arachidonic Acid (ARA) from Arachidonic Acid GlycerineEster

500 g of (commercially available, standard quality) arachidonic acidglycerine ester with an ARA-content of 44.8% % an Anisidine Value of 8.1Ng and a Peroxide Value of 4.0 mmol/was placed in a 3 L flask (renderedinert with nitrogen) and diluted with 500 ml ethanol. 175 g of NaOH(50%) solution and 250 ml water were added and the resulting solutionstirred for 30 min at 30° C. 50° C. Subsequently, the reaction mixturewas diluted with 1250 ml of water and 175 ml sulfuric acid 50 wt-% wasadded thereafter. Phases were separated after 10 min of subsequentstirring and the product phase was washed with 3 time 125 ml water eachand then 125 ml aqueous sodium sulfate solution 10-wt-%. 475 garachidonic acid was obtained as an oil with an Anisidine Value of 4.2Ng and a Peroxide Value of 7.2 mmol/kg. Oligomer content was notdetermined.

Experiment 2: Gamma-Linolenic Acid (GLA) from Gamma-Linolenic Acid EthylEster

500 g of (commercially available, standard quality) gamma-linolenic acidethyl ester with a GC-purity of 99.2%, an Anisidine Value of 19.1 Ng anda Peroxide Value of 52.1 mmol/was placed in a 3 L flask (rendered inertwith nitrogen) and diluted with 500 ml ethanol. 170 g of NaOH (50%)solution and 250 ml water were added and the resulting solution stirredfor 30 min at 30° C. 50° C. Subsequently, the reaction mixture wasdiluted with 1250 ml of water and 175 ml sulfuric acid 50 wt-% was addedthereafter. Phases were separated after 10 min of subsequent stirringand the product phase was washed with 3 time 125 ml water each and then125 ml aqueous sodium sulfate solution 10-wt-%. 500 g gamma-linolenicacid was obtained as an oil with an Anisidine Value of 7.6 Ng and aPeroxide Value of 5.6 mmol/kg. Oligomer content was determined as 0.3area-% (gel-chromatography, RI-detector).

Experiment 3: Arachidonic acid-L-Lysine Salt (ARA-Lys) from ArachidonicAcid (ARA) and L-Lysine) (L-Lys)

70.0 g of arachidonic acid from experiment 1, exhibiting an acid valueof 187 mg KOH/g upon titration, was dissolved in 70.0 g of ethanol andcombined with 34.2 g of an aqueous L-lysine solution (51.3%). Thehomogenous solution obtained after heating to 40-50° C. was spray driedwith a Büchi B190 laboratory-spray drier with an inlet temperature of140° C. and an outlet temperature of 80° C. 81.2 g of a beige powderwith a water content of 0.62% and an ethanol content of <0.1% wereobtained. The salt exhibited an Anisidine Value of 1 Ng and a PeroxideValue of <1 mmol/kg. Oligomer content was determined as 1.1 area-%(gel-chromatography, RI-detector).

Experiment 4: Gamma-Linolenic acid-L-Lysine Salt (GLA-Lys) fromGamma-Linolenic Acid (GLA) and L-lysine) (L-Lys)

70.0 g of gamma-linolenic acid from experiment 2, exhibiting an acidvalue of 137.6 mg KOH/g upon titration, was dissolved in 70.0 g ofethanol and combined with 25.1 g of an aqueous L-lysine solution(51.3%). The homogenous solution obtained after heating to 40-50° C. wasspray dried with a Büchi B190 laboratory-spray drier with an inlettemperature of 140° C. and an outlet temperature of 80° C. 47.9 g of abeige powder with a water content of 1.13% and an ethanol content of<0.1% were obtained. The salt exhibited an Anisidine Value of 5.4 Ng anda Peroxide Value of 1.0 mmol/kg. Oligomer content was determined as 0.6area-% (gel-chromatography, RI-detector).

Experiment 5: Examination of Stability of Arachidonic Acid andGamma-Linolenic Acid and Derivatives Thereof as to the Storage atElevated Temperature (50° C.) and Exposure to Air

30 g each of the liquid ethyl arachidonic acid glycerin ester and thegamma-linolenic acid ethyl ester used in experiments 1 and 2 as well asof the arachidonic acids and the gamma-linolenic acid obtained inexperiments 1 and 2 were filled into 500 ml Schott Duran glass bottles.

About 30 g each of the solid lysine-salts ARA-Lys and GLA-Lys obtainedin experiments 3 and 4 were filled Schott Duran glass bottles.

All of the bottles were placed together with opened lids in a dryingoven with an opened ventilation valve at 50° C. and stored in the darkunder these conditions for 81 days. Results of the analyses performedsubsequently are summarized in the following table (Table 1).

TABLE 1 Anisidine Value Peroxide Value (AV) [A/g] (PV) [mmol/kg]Experiment t = 0 t = 81 days t = 0 t = 81 days ARA- 1 - starting 8.1 2404.0 46.8 glyceride material GLA-OEt 2 - starting 19.1 1237 52.1 86.3material ARA-OH 1 4.2 211 7.2 5.8 GLA-OH 2 7.6 137 5.6 1.3 ARA-Lys 3 1<1 <1 <1 GLA-Lys 4 5.4 <1 1.0 <1

1. A process for increasing the stability towards oxidation of acomposition comprising a polyunsaturated omega-6 fatty acid, saidprocess comprising: (i) providing a starting composition comprising atleast one polyunsaturated omega-6 fatty acid component; (ii) providing alysine composition; (iii) admixing an aqueous, an aqueous-alcoholic oran alcoholic solution of starting composition and lysine composition,and subjecting a resulting admixture to spray drying conditionssubsequently, thus forming a solid product composition comprising atleast one salt of a cation derived from lysine with an anion derivedfrom a polyunsaturated omega-6 fatty acid; the product compositionexhibiting a solvent content SC selected from the following; SC<5 wt %,SC<3 wt %, SC<1 wt %, or SC<0.5 wt %.
 2. The process according to claim1, wherein lysine composition in (ii) is provided in such a manner thatthe ratio R=n(ca)/n(lys) of the amount of carboxylic acid functionsn(ca) in the starting composition provided in (i) and the amount oflysine n(lys) in the lysine composition provided in (ii) is in a rangeselected from 0.9<R<1.1, 0.95<R<1.05, or 0.98<R<1.02.
 3. The processaccording to claim 1, wherein the starting composition provided in (i)does not contain more than x(fe) wt % of fatty acid esters, whereinx(fe) is selected from 5, 3, 2, 1, 0.3, or
 0. 4. The process accordingto claim 1, wherein starting composition in (i) and lysine compositionin (ii) are provided in such a manner that at least sp wt % of theproduct composition consist of one or more salts of cations derived fromlysine with anions derived from one or more polyunsaturated omega-6fatty acids and other naturally occurring fatty acids, wherein sp isselected from 90, 95, 97, 98, 99, or
 100. 5. A composition obtainable bya process according to claim
 1. 6. The process according to claim 1,wherein said starting composition is a food comprising a polyunsaturatedomega-6 fatty acid.
 7. The process according to claim 1, wherein saidstarting composition is a nutritional product comprising apolyunsaturated omega-6 fatty acid.
 8. The process according to claim 1,wherein said starting composition is a pharmaceutical product comprisinga polyunsaturated omega-6 fatty acid.